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WO2008007953A1 - Moyens et procédés pour manipuler la translocation séquentielle phagolysosomique-cytosolique de mycobactéries, et leurs utilisations - Google Patents

Moyens et procédés pour manipuler la translocation séquentielle phagolysosomique-cytosolique de mycobactéries, et leurs utilisations Download PDF

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WO2008007953A1
WO2008007953A1 PCT/NL2007/050323 NL2007050323W WO2008007953A1 WO 2008007953 A1 WO2008007953 A1 WO 2008007953A1 NL 2007050323 W NL2007050323 W NL 2007050323W WO 2008007953 A1 WO2008007953 A1 WO 2008007953A1
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mycobacterium
tuberculosis
espa
esat6
cfplo
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Nicole Neeltje Speelman-Van Der Wel
Michael Barry Brenner
Jacobus Peter Johannes Peters
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Het Nederlands Kanker Instituut
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Het Nederlands Kanker Instituut
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Priority to US12/307,948 priority Critical patent/US20090263418A1/en
Priority to EP07747545A priority patent/EP2046952A1/fr
Publication of WO2008007953A1 publication Critical patent/WO2008007953A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

  • the invention relates to the medical and veterinarian field. More, in particular the invention relates to pathogenesis of mycobacteria and the use of mycobacterial strains as a starting material for vaccines.
  • phagocytes such as macrophages and dendritic cells (DCs) have a significant role in innate host resistance to infection and contribute to the generation of adaptive immune responses.
  • DCs dendritic cells
  • These myeloid cells internalize microbes into membrane bound organelles termed phagosomes that mature and fuse with lysosomes.
  • Phagolysosome fusion creates an acidic environment rich in hydrolytic enzymes that degrade and kill bacteria.
  • proteolysis of bacterial proteins in these compartments generates antigenic peptides that may elicit MHC Class II restricted T cell responses.
  • bacterial evasion strategies targeted at blocking phagolysosome fusion may result in both enhanced survival and delay in the initiation of adaptive immunity.
  • Intracellular pathogens commonly avoid lysosomal fusion through the manipulation of host signal transduction pathways and alteration of endocytic traffic resulting in privileged replicative niches.
  • Salmonella species impede the acquisition of lysosomal hydrolases and reactive oxygen intermediates through the actions of Type III secretion system effector proteins, and reside in an acidified endosome suitable for growth (Waterman and Holden, 2003).
  • Legionella pneumophila induces phagosomes to fuse with secretory vesicles from the ER and Golgi and create an early secretory compartment that is devoid of degradative enzymes and rich in nutrients (Roy and Tilney, 2002; Zamboni et al., 2006).
  • tuberculosis phagosomes fuse with late endocytic multivesicular bodies and lysosomes and at steady-state the bacteria reside in a phagolysosomal compartment. This localization correlates with static bacterial growth over the same time period.
  • a similar phenotype was also detected for M. leprae. Phagolysosomal egression requires live bacteria and does not occur following infection with BCG.
  • the invention provides a method for determining whether a product of a gene of a mycobacterium is involved in translocation of said mycobacterium from the phagosome to the cytosol of a host cell, said method comprising altering said gene product and/or expression of said gene product in said mycobacterium and determining whether said translocation of said mycobacterium in said host cell is affected.
  • Equivalent to altering said gene product and/or expression of said gene product in said mycobacterium is of course to select an already existing mutant mycobacterium wherein said gene product and/or expression of said gene product is altered with respect to the model mycobacterium, preferably the wild type. In this way it is possible to identify genes and gene products that are involved in the translocation to the cytosol.
  • the selected genes or gene products can be promoting the translocation or play a part in inhibiting the translocation. For instance, it has been observed that translocation is a timed process in that it is observed only a few days after infection of the host cell. It has been found that genes and gene products of the specialized secretion system Esx-1 are involved in promoting the translocation. Thus genes and gene products that counteract this secretion system, or the secretion of one or more of the relevant gene products encoded by it, have a repressive effect on translocation and thus promote maintenance of the phagosomal state.
  • said gene is a gene from a region of difference (RD) between mycobacterium tuberculosis and Bacille Calmette Guerin (BCG), or from a corresponding region in another mycobacterium species.
  • BCG is a strain of mycobacterium that is deficient in translocation. It survives and replicates predominantly in the phagosomes of infected cells.
  • BCG is a strain that has been cultured extensively in vitro, and likely as a result of this has lost selected parts of its genome, when compared to wild type species such as mycobacterium bovis and tuberculosis. These selected parts of the genome have been characterised and termed ⁇ regions of difference * .
  • a product of gene for which it is determined whether it is involved in translocation of said mycobacterium from the phagosome to the cytosol of a host cell comprises a product of a gene from a region of difference (RD) between mycobacterium tuberculosis and a Bacille Calmette Guerin (BCG) strain.
  • RD region of difference
  • BCG Bacille Calmette Guerin
  • BCG has effectively been used to immunize humans and particularly juveniles against mycobacterium tuberculosis infection. This is only possible when mycobacterium bovis, and mycobacterium tuberculosis share a large part of their immunogenic epitopes.
  • mycobacterium bovis and mycobacterium tuberculosis share a large part of their immunogenic epitopes.
  • a homologues gene in a non-bovis strain, based on the difference between BCG and bovis.
  • This corresponding gene encodes a gene product that shares at least 90% sequence identity with the RD gene in mycobacterium tuberculosis.
  • Gene products involved in promoting translocation are preferably selected from RDl, from the specialized secretion system Esx-1 and preferably selected from CFPlO, ESAT6 or EspA.
  • Said gene product is preferably a mycobacterium bovis, mycobacterium tuberculosis, kansasii, africanum, leprae, smegmatis or marinum gene product.
  • the gene product can also be a chimeric protein having an amino sequence that is derived from CFPlO, ESAT6 or EspA from two or more mycobacterial strains, or species. Such a consensus CFPlO, ESAT6 or EspA is also part of the invention.
  • the invention further provides a method for reducing the phago-cytosolic translocation of a mycobacterium comprising at least reducing the expression of (consensus) CFPlO, ESAT6 or EspA in said mycobacterium.
  • the expression can be reduced by altering the promoter strength, or it can be reduced by mutating said gene such that the functionality of the gene product is reduced or absent in the thus manipulated mycobacterium.
  • the expression is reduced by deleting the gene encoding CFPlO, ESAT6 or EspA either in whole or in part from the genome. Said part is defined such that the translocation is inhibited.
  • other alterations are within the skill of the person skilled in the art. For instance, frame shift mutations due to insertions are also possible.
  • the invention further provides a method for enhancing phago- cytosolic translocation of a CFPlO, ESAT6 and/or EspA deficient mycobacterium, said method comprising providing said mycobacterium with CFPlO, ESAT6 and/or EspA.
  • a mycobacterium is deficient in CFPlO, ESAT6 and/or EspA when the expression of said product in said mycobacterium is either lacking or suboptimal. It is of course only necessary to provide the gene product that is missing or which presence is suboptimal in said mycobacterium.
  • CFPlO, ESAT6 and EspA a preferred embodiment said bacterium is provided with CFPlO, ESAT6 and/or EspA.
  • said CFPlO, ESAT6 and/or EspA is from the same mycobacterium species as to which it is provided. However, can also be from a different mycobacterium species, or be a consensus CFPlO, ESAT6 and/or EspA. Equivalent to CFPlO, ESAT6 and/or EspA is a protein that shares at least 90% sequence identity with CFPlO, ESAT6 and/or EspA of a mycobacterial species and that shares the same translocation promoting function in kind, not necessarily in amount.
  • Said mycobacterium species is preferably a mycobacterium bovis, mycobacterium tuberculosis, kansasii, africanum, leprae, smegmatis or marinum.
  • Said mycobacterium may also be a strain derived from one of these species, preferably a BCG strain.
  • the invention thus further provides a method for generating a recombinant BCG strain comprising providing BCG or a derivative thereof with CFPlO, ESAT6 and/or EspA.
  • a BCG strain comprising CFPlO, ESAT6 and/or EspA.
  • Such a BCG strain is particularly suited for the preparation of an immunogenic composition as
  • CFPlO, ESAT6 and/or EspA can be provided to said mycobacterium in a number of ways. It is preferably provided by providing said mycobacterium through insertion therein of a nucleic acid encoding CFPlO, ESAT6 and/or EspA.
  • the nucleic acid may be a plasmid or other extrachromosomal nucleic acid.
  • the nucleic acid may be integrated into the chromosomal DNA of said mycobacterium.
  • Said nucleic acid is preferably inserted into said mycobacterium together with the necessary signals for allowing expression of CFPlO, ESAT6 and/or EspA.
  • Said nucleic acid is also possible to insert a coding region in an already present expression cassette.
  • said nucleic acid encoding CFPlO, ESAT6 and/or EspA is provided to said mycobacterium in the absence of at least one other protein coding region of RDl.
  • the invention further provides a recombinant BCG mycobacterium comprising a nucleic acid encoding CFPlO, ESAT6 and/or EspA, a consensus CFPlO, ESAT6 and/or EspA or an equivalent thereof that shares at least 90% sequence identity with CFPlO, ESAT6 and/or EspA of a mycobacterial species and that shares the same translocation promoting function in kind, not necessarily in amount.
  • said mycobacterium is provided with an RDl region, preferably an extended RDl region.
  • a method for producing a mycobacterium that is substantially deficient in phago-cytosolic translocation comprising functionally reducing the expression of CFPlO, ESAT6 and/or EspA in said mycobacterium.
  • Functional reduction of expression is preferably obtained by mutating and/or removing the gene encoding CFPlO, ESAT6 and/or EspA such that substantially no functional CFPlO, ESAT6 and/or EspA is produced by said mycobacterium.
  • a heterologous nucleic acid is inserted into said mycobacterium to reduce said expression.
  • an attenuated mycobacterium comprising a nucleic acid encoding CFPlO, ESAT6 and/or EspA further comprising a heterologous nucleic acid for inhibiting cysolic replication and/or cytosolic translocation of said mycobacterium in a eukaryotic host cell.
  • Said heterologous nucleic acid is preferably present in the genome or a plasmid.
  • Said heterologous nucleic acid is preferably a promoter, preferably a regulatable promoter.
  • Said heterologous nucleic acid is preferably a nucleic acid from another species, preferably not from a mycobacterium species. It is preferred that the expression of said CFPlO, ESAT6 and/or EspA is under transcriptional control of said regulatable promoter. In another preferred embodiment a gene for replication of said mycobacterium is under control of said regulatable promoter.
  • a preferred example of a replication of the invention is dnaA (Greendyke et al (2002) VoI 148: pp 3887-3900).
  • said regulatable promoter is regulatable through the administration of a compound. A preferred but not limiting example is the tet-operon system.
  • the tetracycline system and analogues acting systems have been developed to regulate the action of the promoter by means of a compound that can be added to the surrounding fluid of the cell (see for instance Gossen and Bujard (2002) Annual Review of Genetics. Vol. 36: 153-173.).
  • said mycobacterium further comprises a nucleic acid encoding the transacting factor that dependent on the presence of said compound binds to and regulates said promoter. In this way cysolic replication and/or cytosolic translocation is dependent on the presence or absence of said compound.
  • This property can be used to generate for instance a mycobacterium that is capable of efficient replication and/or translocation in a host thereby enabling the generation of a robust immune response, whereupon the host can be protected from further consequences of the administration by downregulating the expression through the action of the regulatable promoter.
  • Inhibiting cytosolic replication and/or cytosolic translocation impedes the persistence of the infection and allows the host to more easily clear the body from infected cells.
  • Regulatable systems are available that allow expression from the regulatable promoter in the absence or in the presence of said compound.
  • the invention further provides a method for immunizing an individual with a mycobacterium comprising providing said individual with a mycobacterium comprising a heterologous nucleic acid comprising a regulatable promoter of the invention and downregulating expression from said promoter when expression from said promoter is no longer desired.
  • a sufficiently strong immune response has been obtained. This can be achieved by stopping administration of said compound in case of a promoter that is active in the presence of said compound, or by administering said compound in case of a promoter that is active in the absence of said compound.
  • This regulatable system can also be applied to a bacterium of the invention.
  • said foreign antigen comprises a human protein, preferably a human disease associated protein, preferably a tumour associated protein, such as PRAME, MAGE, MUC and 5T4, or mutated or upregulated proteins such as p53 and growth receptors.
  • said foreign antigen comprise a microbial protein or a homologue thereof, preferably a human disease associated viral or bacterial protein, such as HPV, hepatitis, EBV or Helicobacter.
  • said foreign antigen comprises a viral protein or a homologue thereof.
  • said mycobacterium is provided with a nucleic acid encoding said viral antigen or encoding a homologue thereof comprising at least 90% sequence identity with said viral protein.
  • said viral protein is a human virus protein or an animal virus protein.
  • a viral protein from a fish or cow pathogen More preferably said virus comprises a Human Papilloma Virus (HPV), a hepatitis virus or an Epstein-Barr Virus (EBV).
  • HPV Human Papilloma Virus
  • EBV Epstein-Barr Virus
  • ESAT6 and/or EspA to provide a mycobacterium with the capacity to translocate from a phagosome to the cytosol of a host cell, or to enhance said capacity.
  • the invention therefore further provides a method for enhancing and/or inducing cytosolic translocation of a bacterium comprising providing said bacterium with a nucleic acid for expression of CFPlO, ESAT6 and/or EspA of a mycobacterium in said bacterium. Also provided is the use of a nucleic acid for expression of CFPlO, ESAT6 and/or EspA of a mycobacterium in a bacterium for enhancing and/or inducing cytosolic translocation of said bacterium in a eukaryotic host cell. Enhanced and/or induced cytosolic translocation of such bacteria results an enhanced immune response of an individual when exposed to said bacterium.
  • the invention further provides a method for enhancing and/or inducing an MHC-I type related immune response in an individual against an antigen comprising providing a bacterium comprising said antigen with a nucleic acid for expression of CFPlO, ESAT6 and/or EspA of a mycobacterium in said bacterium and administering said bacterium to said individual.
  • said bacterium is not a mycobacterium.
  • said bacterium is a bacterium of a pathogenic bacterial species, preferably a legionella species, preferably I. pneumophila or a salmonella species.
  • said bacterium comprises N. gonorrhoeae or a S. aureus.
  • the invention provides a non mycobacterial bacterium comprising a nucleic acid for expression of CFPlO, ESAT6 and/or EspA of a mycobacterium in said bacterium.
  • a vaccine composition can be administered in various ways.
  • Cell- mediated immunity plays the principal role in containing infection, and the routes of vaccine administration and immunization influences immune response development.
  • BCG vaccination is generally performed by subcutaneous immunization. This generally induces a ThI cytokine response and stimulates cytotoxic T-lymphocyte activity in neonates.
  • An alternative route of BCG administration which does not induce the side effects associated with subcutaneous immunization, is via rectal delivery. This method induces a similar immune response and protection in several animal models without altering the recruitment patterns of activated T-cells.
  • Intranasal immunization induces higher protection by rapid induction of IFN- ⁇ and T-cell response in the lung tissue but there are some who have serious misgivings in using live bacilli.
  • nasal administration of recombinant BCG as a means to deliver immune dominant antigens to the mucosa is possible.
  • the invention provides an animal food or a composition for the production of an animal food comprising a mycobacterium according to the invention, an immunogenic composition according to the invention or a bacterium according to the invention.
  • an oral vaccine comprising a mycobacterium or a bacterium of the invention.
  • mycobacteria such as M. tuberculosis and M. leprae exist in two intracellular sites in human myeloid cells.
  • bacteria reside in a phagolysosome and at extended time points post infection, between 2 and 4 days for M. tuberculosis and between 4 and 7 days for M. leprae the bacilli translocate to the host cytosol.
  • Bacteria in phagosomes rapidly colocalize with the late endosome and lysosomal markers CD63 and LAMP-I and LAMP-2, which are delivered to the phagosome via fusion of multivesicular late endosomes or lysosomes within the first hours of infection.
  • the invention thus further provides a method for infecting host cells with a mycobacterium comprising infecting host cells with said mycobacterium and determining after a period of at least 48 hours and preferably at least 72, more preferably 96 hours, the location of said mycobacterium in said host cells. This is preferably done using microscopy, however, other methods such as flow cytometric, or fractionation approaches are also within the scope of the invention.
  • M. tuberculosis and M. leprae are found in the host cytosol of human DCs and macrophages ( Figure 6).
  • Previous studies showed evidence for cytosolic M. tuberculosis in several cell types including human pneumocytes, rabbit alveolar macrophages, and human monocytes (Myrvik et al., 1984; Leake et al., 1984), however, the prevailing paradigm has remained that M.
  • tuberculosis reside in the endocytic system (Clemens and Horwitz, 1995; Russell, 2001; Russell et al., 2002; Orme, 2004; Vergne et al., 2004; Kang et al., 2005; Pizarro-Cerda and Cossart, 2006).
  • Mycobacterium localization in infected macrophages has been extensively studied for over 40 years using an array of techniques and a number of Mycobacterium species as model organisms for M. tuberculosis. In general, the majority of these experimental systems only focused on the first 48h following mycobacterium infection and were not always performed with virulent mycobacteria. Here we have used an extended time course to examine the localization of M.
  • tuberculosis and M. leprae for up to 7d of infection In our assays, the excellent preservation of cellular membranes in cryosections, coupled with immunological detection of endocytic markers allowed the quantitative assessment of mycobacterial localization to the cytosol at times beyond 2 days of infection.
  • phagolysosomal translocation coincides with an increase in M. tuberculosis titer that continues over the course of the infection.
  • No cytosolic mycobacteria are found after DCs and macrophages phagocytose dead bacteria.
  • the appearance of cytosolic bacteria requires the genes encoded in the ESX-I region, and more specifically the secretion of CFPlO and ESAT6. This finding is further supported by the fact that BCG, which lacks a portion of the ESX-I cluster called the RDl region fails to translocate into the cytosol and remains localized to the phagolysosome. In addition to M.
  • L. monocytogenes that lyse host phagosomes and replicate in the host cytosol induce potent CD8+ T cell responses. Lysis of the phagosomal membrane requires the cholesterol dependent cytolysin Listeriolysin O (LLO), which has a slightly acidic pH optimum and a short-half life in the host cytosol (Glomski et al., 2002; Schnupf et al., 2006; Decatur and Portnoy, 2000).
  • LLO cholesterol dependent cytolysin Listeriolysin O
  • M. marinum has been shown to escape from phagosomes in infected macrophages and spread to neighbouring cells via actin based motility (Stamm et al., 2003; Stamm et al., 2005). It is noteworthy that in a Rana pipiens model of long-term granuloma formation, 60% of M. marium phagosomes were fused with lysosomes (Bouley et al., 2001). Therefore, it seems likely that M. tuberculosis has evolved additional mechanisms of immune escape that allow survival when the blockade of phagosome-lysosome fusion is overcome by the host. These might be significant at later stage of infection or upon cytokine activation of infected antigen presenting cells.
  • CD4+ T cells dominate during acute infection and CD8+ T cells during persistent infection (Lazarevic et al, 2005). How antigens from intracellular bacteria gain access to the MHC Class I antigen loading pathway in the ER remains an intense area of study.
  • BCG which is used worldwide as a mycobacterial vaccine strain remains restricted to the phagolysosome following infection of DCs and macrophages, whereas virulent M. tuberculosis does not (Figure 6).
  • BCG vaccination has questionable efficacy against the highly infectious pulmonary form of tuberculosis, and it fails to generate a strong MHC class I restricted T cell response.
  • the work presented here emphasizes that non-virulent mycobacterial species fail to translocation the phagosome and suggests this may account for their poor capacity to stimulate critical CD8+ T cell responses.
  • innovative vaccine approaches have genetically engineered BCG to express LLO as a mechanism to generate more potent MHC Class I-restricted responses.
  • LLO+ BCG are more effective vaccines than the isogenic BCG parental strain (Grode et al., 2005). Designing vaccines that mimic virulent strains in translocating into the cytosol is likely to be a critical step forward in producing more effective vaccines for tuberculosis. Examples
  • M. tuberculosis and M. leprae reside in a phagolysosome early after phagocytosis
  • M. tuberculosis and M. leprae The subcellular localization of M. tuberculosis and M. leprae was analyzed in freshly isolated human monocyte-derived DCs.
  • Monocyte-derived DCs were differentiated from human CD 14+ monocytes precursors for 5 days in GMCSF and IL-4, and subsequently infected with M. tuberculosis or M. leprae. Samples were fixed at various times after infection (8 min to 48h) and processed for cryo-immunogold electron microscopy.
  • the phagosome lacked the early endosomal markers transferrin receptor (TfR) and early endosomal autoantigen 1 (EEAl), which instead were exclusively localized to early endocytic and recycling endosome membranes (Table 1).
  • TfR transferrin receptor
  • EAAl early endosomal autoantigen 1
  • the phagosome was also negative for the late endosomal cation-independent mannose 6-phosphate receptor (Table 1).
  • both M. tuberculosis and M. leprae phagosomal membranes stained positively for the lysosomal associated membrane proteins LAMP-I, LAMP-2, and CD63 ( Figure IA-F and Table 1).
  • MIIC MHC class II compartment
  • LAMP-I and LAMP-2 localized on the limiting membrane and CD63 on internal membranes.
  • MDL DC lysosome
  • M. tuberculosis access the host cytosol and replicate
  • monocyte-derived DCs were infected with M. tuberculosis and plated in replicate wells of a 24-well plate. At each time point, DCs were lysed and the number of colony forming units (CFU) per well was enumerated.
  • CFU colony forming units
  • M. tuberculosis persist during the initial 48 h infection period in monocyte-derived DCs, but are able to replicate significantly only after that time point.
  • the increase in bacterial titer between day 2 and 3 suggested that alterations occur to the phagolysosome that create a more favourable growth environment.
  • monocyte-derived DCs infected with M. tuberculosis were fixed and processed for cryo immuno-gold labelling with anti-LAMP-1 antibody at 48 and 96h. As at the earlier time points, M.
  • bacteria were found that lacked the characteristic electron lucent zone (Armstrong and Hart, 1971) and did not stain positively for LAMP-I ( Figure 2B).
  • Figure 2B the characteristic electron lucent zone
  • these bacteria were not present in membrane enclosed compartments and appeared to be localized to the cytosol.
  • bacteria only partially surrounded by phagolysosomal membranes were seen and may represent bacteria at an intermediate stage of translocation from the phagolysosome ( Figure 2B arrowhead).
  • tuberculosis and BCG identified several large deletions from BCG that are present in M. tuberculosis and M. leprae (Harboe et al., 1996; Gordon et al, 1999; Behr et al., 1999; Philipp et al., 1996). From these 16 regions of difference (RD1-16) only RDl is absent from all BCG strains thus far tested (Mostowy et al., 2002; Tekaia et al., 1999; Brosch et al., 2002). RDl is part of a 15-gene locus known as ESX-I that encodes a specialized secretion system dedicated to the secretion of CFPlO and ESAT6.
  • ESX-I 15-gene locus
  • PBMC Peripheral blood mononuclear cells
  • Immature monocyte-derived DCs were prepared from CD14+ monocytes by culture in 300 U/ml of granulocyte - macrophage colony- stimulating factor (GM-CSF, Sargramostim, Immunex, Seattle, WA) and 200 U/ml of IL-4 (PeproTech, Rocky Hill, NJ) for 5d in complete RPMI medium (10% heat-inactivated FCS/20mM Hepes/2mM L- glutamine/lmM sodium pyruvate/55 ⁇ M 2-mercaptoethanol/Essential and nonessential amino acids). GMCSF and IL4 were replenished on d2, d5, and d9 after isolation. Macrophages were prepared by culture of CD 14+ monocytes in IMDM with 10% human AB serum, 2mM L-glutamine, and 50ng/mL M-CSF (PeproTech, Rocky Hill, NJ).
  • GM-CSF granulocyte - macrophage colony- stimulating factor
  • IL-4 PeproTech
  • M. tuberculosis strains were grown to mid-logarithmic phase from frozen stocks in 7H9 Middlebrook media containing OADC enrichment solution and 0.05% Tween-20 for 1 week at 37 0 C.
  • the wild-type M. tuberculosis strain used in these studies was H37Rv expressing green fluorescent protein (GFP) (Ramakrishnan et al., 2000).
  • the BCG Pasteur strain was provided by Barry Bloom.
  • the Tn::Rv3874 (cfplO) and the DespA strain have been previously described (Guinn et al., 2004a; Fortune et al., 2005).
  • the DespA strain complemented strain encodes espA under the control of its native promoter on an integrating vector.
  • the construct has been shown to complement the DespA mutation for ESAT6 secretion (S. Fortune, Personal communication).
  • M. leprae were purified from mouse footpads as previously described and used in experiments one day after isolation (Adam
  • the LAMP-I labelling density number of gold particles per ⁇ m phagosomal membrane (LD) as determined on at least 30 phagolysosomes in DCs infected with M. tuberculosis for 2, 24, 48 hours, and 48 h (M. leprae is included for the last time point) and compared to the LD on the limiting membrane of lysosomes or the background labelling on mitochondria in the same cells.
  • LD phagosomal membrane
  • A The number of M. tuberculosis per infected DC at 4, 24, 48, 96 hours after infection in different subcellular compartments.
  • the phagolysosomal mycobacteria are characterized by enclosure of a LAMP-I labelled membrane and the cytosolic bacteria lack both a membrane and LAMP-I labelling. Data shown is based on at least 30 cells per time point and is a representative result out of 5 experiments.
  • B The number of live or heat killed M. tuberculosis in macrophages and DCs infected for 96 hours.
  • M. bovis BCG does not translocate from the phagolysosome
  • A The number of M. bovis BCG per infected DC at 2,4 and 7 days in different subcellular compartments. The number of bacteria as determined in LAMP-I labelled membrane enclosed compartments denoted as phagolysosomes, in phagosome defined as membrane enclosed compartments lacking LAMP-I and in compartments lacking both membrane and LAMP-I labelling defined as the cytosol.
  • B The colony forming units (CFU) determined for M. bovis BCG infected DCs. Multiple experiments from which a representative figure is shown, all demonstrated that the CFU increases over time, suggesting that replication occurs.
  • C Representative EM image of DC infected with M.bovis BCG for 7 days and immunogold labelled against LAMP-I. Asterisks indicate phagolysosomal M.bovis BCG , L: lysosomes, M: mitochondria, N: nucleus, ER: endoplasmic reticulum, bar: 200 nm.
  • M. tuberculosis RDl mutants do not translocate from the phagolysosome
  • tuberculosis ⁇ espA tuberculosis ⁇ espA for 7 days; immunogold labelled for LAMP-I demonstrates that M. tuberculosis ⁇ espA remains in a membrane enclosed LAMP labelled compartment. Asterisks indicate phagolysosomal M. tuberculosis ⁇ espA, L: lysosomes, M : mitochondria and bar: 200 nm, Figure 6
  • Initial host-pathogen encounters include bacterial interactions with epithelial tissues that serve as physical barriers to invasion and infection.
  • host phagocytes and antigen presenting cells such as macrophages and dendritic cells (DCs) have a significant role in innate host resistance to infection and contribute to the generation of adaptive immune responses.
  • DCs dendritic cells
  • myeloid cells internalize microbes into membrane bound organelles termed phagosomes that mature and fuse with lysosomes. Phagolysosome fusion creates an acidic environment rich in hydrolytic enzymes that degrade and kill bacteria.
  • proteolysis of bacteria in these compartments generates antigens that may elicit MHC or CDl restricted T cell responses.
  • Intracellular pathogens commonly avoid lysosomal fusion through the manipulation of host signal transduction pathways and alteration of endocytic traffic resulting in privileged replicative niches.
  • Listeria monocytogenes and Shigella flexneri lyse the phagosomal membrane and escape from the endocytic system into the host cytosol where they replicate and are able to spread to neighboring cells via actin-based motility (Stevens et al., 2006).
  • Nearly all intracellular pathogens have specialized to manage their fates as "endosomal" or "cytosolic" pathogens.
  • M. tuberculosis and M. leprae reside in a phagolysosome early after phagocytosis
  • the subcellular localization of M. tuberculosis and M. leprae was analyzed in freshly isolated human monocyte-derived DCs.
  • DCs were differentiated from human CD 14+ monocytes precursors for 5 days in GM-CSF and IL-4, and subsequently infected with M. tuberculosis H37Rv or M. leprae. Samples were fixed at various times after infection (2-48 hours) and processed for cryo-immunogold electron microscopy (Peters et al., 2006). We analyzed the localization of early and late endosomal markers to the M. tuberculosis or M.leprae phagosome.
  • the phagosome lacked the early endosomal markers transferrin receptor (TfR) and early endosomal autoantigen 1 (EEAl), which instead were exclusively localized to early endocytic and recycling endosome membranes (Table 1).
  • TfR transferrin receptor
  • EAAl early endosomal autoantigen 1
  • the phagosome was also negative for the late endosomal cation-independent mannose 6-phosphate receptor (Table 1).
  • both M. tuberculosis and M. leprae phagosomal membranes labelled for the lysosomal associated membrane proteins LAMP-I, LAMP-2, CD63 and the major lysosomal aspartic proteinase cathepsin D ( Figure IA-D and Table 1).
  • the mycobacteria reside in a compartment that readily fuses with lysosomes and forms independent of the ER.
  • tuberculosis (Figure 6B, D), demonstrating a role for the ESX-I system and the secretion of CFP-10 and ESAT-6 in the translocation of JVf. tuberculosis from the host endocytic system.
  • FtsZ a bacterial tubulin like protein.
  • FtsZ is critical for the cell division process in many prokaryotes including mycobacteria and is transiently higher expressed during cytokinesis (Margolin, 2005).
  • the relative immunogold labelling index for FtsZ was determined on mycobacteria in the cytosol and in phagolysosomal compartments at different times of infection and compared to the labelling on cellular compartments as control (supplementary Figure 4). The data demonstrate at 7 days of infection the highest amount of FtsZ in cytosolic JVf.
  • tuberculosis relative to phagolysosomal bacteria suggesting that JVf. tuberculosis preferably replicates in the cytosol.
  • Tn::CFP-10 mutant replicates in the phagolysosomal compartments.
  • the RDl locus is also present in M. bovis, M. kansasii, M. marinum, M. africanum, and M. leprae (Berthet et al., 1998; Harboe et al., 1996).
  • the ESX-I region has an important role in the virulence of M. tuberculosis (Lewis et al., 2003; Hsu et al., 2003; Stanley et al., 2003).
  • the genes encoded in the ESX-I region are predicted to form a specialized secretory apparatus that secretes CFP-IO and ESAT-6. Pathogens such as L.
  • leprae antigens presented by MHC class I are most likely derived from bacteria that have entered the host cytosol as shown here (see Figure 7B).
  • Recent in vivo work (Majlessi et al., 2005) and unpublished data presented at the TB Keystone meeting 2007 confirm this suggestion by showing a significant increase of MHC class I restricted CD8+ T cell response in a recombinant BCG strain in which the extended RDl region is introduced (R. Billeskov and J. Dietrich, personal communication) or by showing that the T cell response to CFP-IO and ESAT-6 is eliminated in M. tuberculosis mutations affecting the function of the ESX-I secretion system (S. Behar, personal communication)
  • PBMC Peripheral blood mononuclear cells
  • the AespA strain complemented strain encodes espA under the control of its native promoter on an integrating vector (delta3616+p3616).
  • the construct has been shown to complement the AespA mutation for ESAT-6 secretion (S. Fortune, Personal communication).
  • M. leprae were purified from mouse footpads as previously described and used in experiments one day after isolation (Adams et al., 2002).
  • cells were fixed by adding an equal volume of 2x fixative (0.2M PHEM buffer containing 4% paraformaldehyde or 0.4% Gluteraldehyde and 4% paraformaldehyde) to plates immediately after removal from the incubator. Cells were fixed for 24 hours at room temperature and recovered using a cell scraper. Fixed cells were stored in 0.1M PHEM buffer and 0.5% paraformaldehyde until analysis. Fixed cells were collected, embedded in gelatine and cryosectioned with a Leica FCS and immuno labelled as described previously (Peters et al., 2006).
  • 2x fixative 0.2M PHEM buffer containing 4% paraformaldehyde or 0.4% Gluteraldehyde and 4% paraformaldehyde
  • MHC class I Hsu
  • FtsZ a gift from Dr. Rajagopalan
  • TAP 198.3 a gift from Dr. J. Neefjes
  • PDI a gift from Dr. H. Ploegh
  • Antibodies were labelled with rabbit anti- mouse bridging serum (DAKO) and protein-A conjugated to 10 nm gold (EM laboratory, Utrecht University). Sections were examined using a FEI Tecnai 12 transmission electron microscope. Quantitation was done according to routine stereological methods. The labelling density and relative labelling index determined for respectively MHC class I and FtsZ was calculated according to Mayhew (Mayhew et al., 2002)
  • LAMP-I labelling density number of gold particles per ⁇ m phagosomal membrane as determined on at least 30 phagolysosomes in DCs infected with M. tuberculosis for 2, 24 and 48 hours, and M. leprae 48 hours remains equal and compared to the LD on the limiting membrane of lysosomes (L) slightly lower.
  • L LAMP-I labelling density
  • Fluorescence image of DCs infected with M. tuberculosis (green) for 4 hours labelled with anti cathepsin D (red) or LAMP-I (red) and DAPI (blue) demonstrates that at early stages the bacteria are present in a phagolysosomal compartment. Merged images on the right panel.
  • D No co-localisation of M. tuberculosis with LAMP-I and cathepsin D after 96 hours Fluorescence images of DCs infected for 96 hours in which large clusters of M tuberculosis (green) bacteria are present. Most of these clusters do not co-localize with the lysosomal markers cathepsin D (red) and LAMP-I (red) although individual bacteria were shown (arrow head) to co- localise. Merged images on the right panel.
  • Electron micrograph of a DC infected with M. tuberculosis for 48 hours showing different subcellular locations: 1) mycobacteria observed in membrane-enclosed phagolysosomes (asterisk) which are characterized by an electron lucent zone between the phagosomal membrane and the bacterial cell wall and immunogold labelling with LAMP-I on the phagolysosomal membrane. 2) mycobacteria detected in the cytosol (encircled asterisk) lacking the enclosure of a membrane and the LAMP-I labelling (more examples in Figure: 3B, 6D and supplemental Figure 2 and 3B).
  • Layers in the bacteria are identical to the cytosolic layers with the addition of two cellular layers: d) phagosomal or electron lucent space, which varies in size, e) phagosomal membrane, immunogold labelled for LAMP-I.
  • L lysosomes
  • M mitochondrium
  • asterisk mycobacteria in phagolysosomes
  • encircled asterisks cytosolic mycobacteria. All images are from cryo- immunogold labelled cryo-sections. Bar: A) 300 nm, B) 500 nm.
  • the reconstruction was made from a -60 degree to +60 degree tilt series taken in 1 degree increments.
  • the reconstruction was made using weighted back projection using the IMOD software.
  • the specimens were sectioned in thick (200 nm) sections to enlarge the chance of including membranous structures however; no membranes surrounding the bacteria were detected. Movie available in Supplementary Figure 2D. Encircled asterisk: cytosolic M. tuberculosis, M: mitochondrium, L: lysosome.
  • M. leprae per infected DC as observed on immunogold EM labelled cryo-sections at day 4 and 7 in phagolysosomes, phagosomes and in the cytosol.
  • the phagolysosomal, phagosomes and cytosolic mycobacteria are characterised as described in Figure 3A. Error bars represent standard errors.
  • M. leprae resides in all compartments.
  • CFU colony forming units
  • the number of bacteria was determined as described for Figure 3A.
  • the espA deletion mutant does not translocate while the complemented espA mutant (deta3616+p3616) and the wild type Af. tuberculosis H37Rv (Mtb) translocate to the cytosol.
  • MHC class I molecules are not present on the phagolysosome. (A) Low labelling density of MHC class I on phagosomes
  • L lysosomes
  • M mitochondrium
  • asterisk mycobacteria in phagolysosomes
  • encircled asterisks cytosolic mycobacteria. All images are from cryo- immunogold labelled cryo-sections. Bar: C) 100 nm and D) 200 nm.
  • FtsZ protein demonstrates replication of cytosolic M. tuberculosis
  • DCs infected with M. tuberculosis or M. tuberculosis Tn were immunogold labelled for FtsZ.
  • the amount of gold particles relative to the surface size of bacteria in phagolysosomes, bacteria in cytosol, mitochondria (mito), lysosomes (lyso) and nucleus was determined and the relative labelling index (RLI) calculated according to Mayhew (Mayhew et al., 2002).
  • the bacteria in the cytosol contain increased amounts of FtsZ, demonstrating increased replication of M. tuberculosis in the cytosol, while the CFP-10 mutant of M. tuberculosis replicates in the phagosome.
  • Epon embedded DCs infected with M. tuberculosis Epon embedded DCs infected with M. tuberculosis
  • the Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells.
  • ER-phagosome fusion defines an MHC class I cross- presentation compartment in dendritic cells. Nature 425, 397-402. Guinn,K.M., Hickey,M.J., Mathur,S.K., Zakel,K.L., Grotzke,J.E.,
  • Phagosomes are competent organelles for antigen cross-presentation. Nature 425, 402-406.
  • Mycobacterium bovis BCG-immune alveolar macrophages are resistant to disruption by Mycobacterium tuberculosis H37Rv. Infect Immun 45, 443- 446.
  • CDIb restricts the response of human CD4-8- T lymphocytes to a microbial antigen. Nature 360, 593-

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Abstract

Les Mycobactéries, telles que les M. tuberculosis et M. leprae, sont considérées comme des bacilles intracellulaires prototypiques, qui ont des stratégies développées pour permettre leur croissance dans les phagosomes intracellulaires de la cellule hôte. Il est démontré au contraire que les lysosomes fusionnent rapidement avec les phagosomes contenant des M. tuberculosis et M. leprae virulents de cellules dendritiques dérivées de monocytes humains et de macrophages. Après 2 jours, les M. tuberculosis subissent une translocation progressive des phagolysosomes au cytosol, où ils se répliquent. L'entrée cytosolique est également observée pour le M. leprae, mais pas pour la souche vaccinale, le M. bovis BCG, ou des mycobactéries tuées, et celle-ci est dépendante de la sécrétion des produits géniques mycobactériens CFP-IO et ESAT-6 de la région RD1. La présente invention procure en outre des moyens et procédés pour utiliser ces découvertes dans des compositions thérapeutiques et immunogènes.
PCT/NL2007/050323 2006-07-10 2007-06-29 Moyens et procédés pour manipuler la translocation séquentielle phagolysosomique-cytosolique de mycobactéries, et leurs utilisations Ceased WO2008007953A1 (fr)

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